Pancreatic secretion of digestive enzymes plays an essential role in the digestion of food. Digestive enzymes synthesized by acinar cells are packaged into secretory granules termed zymogen granules. Upon stimulation of the pancreas by gastrointestinal hormones and neurotransmitters the zymogen granules fuse with the luminal membrane of the acinar cell such that the granule contents are released. Little is known about this process at the molecular level. Because protein-protein interactions are almost certainly involved in the fusion of the zymogen granule membrane with the plasma membrane it is essential to identify and characterize zymogen granule membrane proteins. Zymogen granules can be highly purified on Percoll gradients, their membranes isolated and the component proteins resolved by 2-dimensional gel electrophoresis. Proteins will be characterized by probing blots of gels with stains, antibodies, lectins and 35S-GTP. Preliminary work has shown multiple yet unidentified small G proteins firmly associated with the granule membrane. The topology of the proteins will be studied by biotinylating or otherwise labeling the outside of intact granules prior to lysis and in the intact cell using Ab against component proteins. N-terminal microsequence will be obtained from blots and used to predict nucleotide sequence to clone by PCR and/or library screening. Preliminary work has obtained partial amino acid and nucleotide sequence for a glycoprotein termed GP-3. Antibodies will then be generated to synthetic peptides or cloned proteins expressed in E. Coli. Ab and cDNA probe will be used for immunocytochemistry to confirm granule membrane localization and to establish whether the protein is unique to pancreas or common to other secretory granule membranes. Ab or synthetic peptide sequences to proteins on the external face of the granule will be evaluated in permeabilized pancreatic acini for their ability to inhibit secretion. Pancreatic AR42J cells and AtL-20 cells will be transfected with sense and antisense constructs to regulate expression of granule proteins and evaluate effects on packaging of granule contents and secretion.